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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2760-2771
Identification of a Common Developmental Pathway for Thymic Natural
Killer Cells and Dendritic Cells
By
Carlos Márquez,
César Trigueros,
Jaime M. Franco,
Almudena R. Ramiro,
Yolanda R. Carrasco,
Miguel López-Botet, and
María L. Toribio
From the Centro de Biología Molecular "Severo Ochoa,"
Universidad Autónoma de Madrid, Madrid, Spain; and the Servicio
de Inmunología, Hospital de la Princesa, Madrid, Spain.
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ABSTRACT |
Current data support the notion that the thymus is seeded by a yet
uncommitted progenitor cell able to generate T cells, B cells, natural
killer (NK) cells, and dendritic cells (DCs). We assess in this report
the developmental relationship of DCs and NK cells derived from a small
subset of CD34+ human postnatal thymocytes that, like the
earliest precursors in the fetal thymus, display low CD33 surface
expression. Culture of these isolated CD34+
CD33lo thymic progenitors with a mixture of cytokines,
including interleukin-7 (IL-7), IL-1 , IL-6, granulocyte-macrophage
colony-stimulating factor, and stem cell factor, results in predominant
generation of DCs. However, the addition of IL-2 to the cytokine
mixture leads to the simultaneous development of DCs and NK
cells. Both developmental pathways progress through a transient
population of CD34+CD44bright
CD5lo/ CD33+ large-sized cells,
distinct from small-sized T-lineage precursors, that contain
bipotential NK/DC progenitors. These data provide evidence of linked
pathways of NK cell and DC development from intrathymic precursors and
suggest that NK cells and DCs branch off the T lineage through a common
intermediate progenitor.
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INTRODUCTION |
ALL LYMPHOID CELL types derive,
ultimately, from pluripotent hematopoietic stem cells (HSCs) that
reside in the liver during embryonic development and then in the bone
marrow during the postnatal life.1 A major issue is whether
commitment of HSCs into a given lymphoid lineage is an abrupt process
or whether there exist oligopotent intermediate progenitors common to
all lymphoid lineages, but no longer able to generate myeloid,
erythroid, or megakaryocytic cells.2,3 Although the
existence of a common lymphoid progenitor has been postulated for
years,4 only very recently has formal proof been provided
by Galy et al5 that such a cell type does exist in the
human bone marrow. This cell population represents a phenotypically
defined subset of CD34+ bone marrow cells
(CD34+CD10+CD45RA+CD38+HLA-DR+)
that is distinct from CD34+ HSCs and has a unique
differentiation potential restricted to the production of all lymphoid
cells (T, B, and natural killer [NK] cells) and also of dendritic
cells (DCs). The identification of such a common lymphoid/DC progenitor
challenges current views on the myeloid lineage affiliation of most
DCs6-8 and supports the notion that the DC lineage, or at
least a particular lineage of DCs, is developmentally more closely
related to lymphoid cells than to myeloid cells.
This appears to be the case for the so-called thymic DCs. In fact, the
earliest mouse intrathymic precursors that are capable of forming T, B,
and NK cells also serve as precursors of DCs on transfer to irradiated
recipients, without concomitant formation of erythroid or myeloid
cells.9-13 This situation can be extended to humans, in
whom equivalent lymphoid/DC progenitors have been identified both in
the fetal and in the postnatal thymus and have also been found in the
fetal liver.14-17 As a whole, the hematopoietic developmental potential of the earliest intrathymic progenitor pool
matches that of the lymphoid/DC-restricted progenitor identified in the
bone marrow, suggesting that such a oligopotent precursor subset might
include the initial cells that migrate out of bone marrow to seed the
thymus.3,18
Little is known about the critical points of lineage decision leading
such intrathymic lymphoid/DC precursors to commit to each alternative
(T, B, NK, and dendritic) lymphoid cell lineage. Intrathymic precursors
in mice had shown a sequential rather than simultaneous loss of other
developmental potentials en route to T cells, suggesting that
non-T-cell lineages stem from distinct branch points in the
differentiation pathway of a common progenitor. Particularly, it has
been shown that the capacity to form B cells is lost in the murine
thymus in a T-cell precursor subset that still retains a full potential
to generate DCs19 and also forms NK cells, albeit with
lower efficiency than its immediate progenitor,13 indicating that this precursor population must be
tripotential.18 In addition, a formal clonal proof has been
provided that human T cells and NK cells share a common intrathymic
precursor,15 leading to the current view that the branching
of NK and T cells occurs downstream of the divergence of
DCs.20 However, it is not known whether such a T/NK
bipotential progenitor can also derive DCs; therefore, no formal
evidence has been produced that DCs branch off the main T-cell
developmental pathway before NK cells. We now assess the developmental
relationship of thymic DCs and NK cells derived from the most immature
CD34+CD33lo human thymocyte precursors. Our
data provide evidence of linked pathways of NK cell and DC development
from intrathymic precursors in vitro and suggest that NK cells and DCs
branch off the T lineage through a common intermediate bipotential
progenitor.
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MATERIALS AND METHODS |
Isolation of subpopulations of human postnatal thymocytes.
Normal human postnatal thymocytes were isolated from thymus fragments
removed during corrective cardiac surgery of patients aged 1 month to 4 years.14 Thymocyte suspensions were enriched in immature
thymocytes by using the sheep red blood cell (SRBC) rosetting technique
described by Poggi et al.21 Recovered cells (1% to 2% of
cellular input, 30% to 60% CD34+) were depleted of B,
myeloid, and NK cells by treatment with anti-CD19-coated magnetic
beads (Dynabeads; Dynal, Oslo, Norway) and anti-CD14 and anti-CD56
(Becton Dickinson & Co, San José, CA) monoclonal antibodies
(MoAbs) bound to sheep antimouse Ig-coated magnetic beads (Dynal). To
isolate CD34+CD1 thymocytes,
CD1+ cells were first removed by treatment with anti-CD1a
(OKT6, CRL8020; American Type Culture Collection [ATCC],
Gaithersburg, MD) MoAbs bound to magnetic beads (Dynal) and then cells
expressing intermediate to bright levels of CD34
(CD34int-bright) were positively isolated with the Dynal
CD34 Progenitor Cell Selection System following the manufacturer's
instructions. The isolated cell preparation (0.1% to 0.25% of total
thymocytes) was highly enriched in CD34+
CD1 thymocytes (>98%). Twenty percent to 30% of
these cells expressed low levels of CD33. These
CD34+CD1CD33lo cells were
then isolated by cell sorting (see below). In some experiments,
thymocyte preparations depleted of B, myeloid, and NK cells (as
described above) were also depleted of mature T cells by treatment with
anti-CD3- and anti-CD8-coated magnetic beads (Dynal). Such
lin (CD19,
CD14, CD56,
CD3) thymocytes were finally treated with the CD34
Progenitor Cell Selection System (Dynal), without previous treatment
with anti-CD1a. The remaining cellular population was shown to include
two distict subsets of thymocytes expressing low CD34 levels, one
CD44brightCD5lo/CD33+ and
the other CD44loCD5+CD33.
Sheep defibrinated erythrocytes in Alsever solution were purchased from
Unipath Ltd (Hampshire, UK).
Immunofluorescence, flow cytometry, and cell sorting.
MoAbs against the following antigens were used: CD2 (T11-RD1), CD11b
(MO1-fluorescein isothiocyanate [FITC]), CD14
(MO2-FITC), and CD25 (IL2-R1-FITC) from Coulter Clone (Hialeah, FL);
CD1a (T6-RD1 [from Coulter Clone] and OKT6 [from the ATCC]), CD34
(CD34-phycoerythrin [PE]-Cy5 [from Immunotech, Marseille, France]
and HPCA-2-PE [from Becton Dickinson & Co]); CD3 (Leu-4-PE), CD5
(Leu-1-PE), CD8 (Leu-2a-FITC), CD13 (Leu-M7-PE), CD19 (Leu-12-FITC),
CD22 (Leu-14-PE), CD38 (Leu-17-PE), CD80 (B7-BB1), and HLA-DR
(anti-HLA-DR-PE) from Becton Dickinson & Co; CD44 (CD44-FITC), CD4
(CD4-Tri-Color), and CD11c (CD11c-FITC), from Caltag Laboratories
(South San Francisco, CA); CD33 (Leu-M9-PE [from Becton Dickinson & Co] and CD33 biotin-conjugated [from Caltag]); CD7 (Leu 9-FITC
[from Becton Dickinson & Co] and T3-3A1 and HB 2 [from the ATCC]);
CD40 (MoAb89 [kindly provided by Dr J. Banchereau, Schering-Plough,
Dardilly, France] and CD40-FITC [from Caltag]); CD86 (CD86-FITC
[from Serotec, Oxford, UK]); CD56 (Leu-19-PE [from Becton Dickinson
& Co] and CD56-Tri-Color [from Caltag]); and CD122 (DU-2) from
Olimpus Corp Immunochemicals (New York, NY). The
anti-CD161 specificity (anti-NKR-P1A) of HP-3G10 MoAb was assigned at
the Sixth International Workshop on Human Leukocyte Differentiation
Antigens. The anti-CD45RA MoAb was generously provided by Dr J.C.
Gutiérrez-Ramos (Millennium Pharmaceuticals Inc, and Department
of Medicine, Boston University Medical Center, Boston, MA) and the
anti-HLA-DP and -DQ MoAbs were the kind gift of Dr J. Bodmer (Imperial
Cancer Research Fund, London, UK).
Single-, two-, or three-color immunofluorescence stainings were
performed on cells previously incubated with PBS/EDTA buffer for 15 minutes, as described elsewhere.14 Second-step reagents including FITC-, PE-, or PE-Cy5-conjugated goat antimouse
F(ab2) IgGs or IgG1 and PE- or PE-Cy5-conjugated
streptavidine were purchased from Caltag. Stained cells were analyzed
in a flow cytometer (EPICS Profile; Coulter Electronics Inc, Hialeah,
FL). Data were collected on 1 to 3 × 104 viable cells
as determined by electronic gating on forward and side scatter light
parameters. Isotype-matched irrelevant antibodies from Caltag were used
as negative controls to define background fluorescence. Cell sorting of
CD34+CD44+CD33 and
CD34+CD44+CD33lo thymocytes was
performed with a FACStar plus (Becton Dickinson & Co) on isolated
CD34+CD1 thymocytes after labeling with
anti-CD44-FITC, anti-CD33-PE, and anti-CD34-PE-Cy5. Sorted cells were
95% to 98% pure as determined by post-sort analysis.
Cell cultures.
Either CD34+CD1CD33
or CD34+CD1CD33lo thymocytes
(5 × 105 cells/mL) were cultured in 96-well
flat-bottomed microtiter plates (Costar, Cambridge, MA) in RPMI 1640 medium (GIBCO, Paisley, UK) supplemented with 10% fetal calf serum
(FCS; GIBCO), in the presence of 100 International Units (IU)/mL
recombinant human interleukin-7 (rhIL-7), 60 IU/mL rhIL-1, 100 IU/mL
recombinant human stem cell factor (rhSCF), 50 IU/mL rhIL-6, and 75 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; from
the National Institute of Biological Standards and Controls, Potters
Bar, Hertfordshire, UK). When indicated, rhIL-2 (from Hoffman La Roche,
Basel, Switzerland) was added to the multicytokine-supported cultures
at doses favoring high-affinity IL-2 receptor binding (10 IU/mL, 40 pmol/L). When used singly, IL-2 and IL-7 were used at 200 IU/mL and
1000 IU/mL, respectively (Table 1).
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Table 1.
Absolute Frequencies of NK Cells and DCs Generated From
CD34+CD33lo Postnatal Thymocytes in Different
Culture Conditions
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Limiting dilution analysis.
CD34+CD1CD33lo thymocytes
were diluted in RPMI 1640 medium supplemented with 10% FCS in the
presence of the cytokine mixture described above either with or without
IL-2 (200 IU/mL). Cells were plated at 300, 100, 33, 10, 3, and 1 cell
per well onto Terasaki microplates (Costar; 1 plate per cellular
dilution) and cultured for 16 days. Cytokine-supplemented medium was
changed every 6 to 7 days by demidepletion. In some experiments, cells
were first maintained during 3 or 4 days under high-density culture
conditions (5 × 105 cells/mL), as described above, in
the presence of the cytokine mixture without IL-2. Afterwards, cells
were plated by limiting dilution and cultured in the presence of the
cytokine mixture either with or without IL-2 for 16 days. At the end of
the culture, all wells were microscopically inspected, and the number
of wells containing cells with a typical dendritic morphology (veiled
cells and cells with dendritic processes) was scored. The same wells were analyzed for the presence of CD56+ cells after
incubation for 30 minutes at 4°C with anti-CD56 (Leu-19; Becton
Dickinson & Co) MoAbs bound to goat antimouse IgG-coupled magnetic
beads (Dynal). Wells containing cells coated with magnetic particles
were scored as positive. The maximum likelihood estimate of clonogenic
precursors was calculated with the single hit Poisson model.22
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RESULTS |
CD34+CD33lo postnatal thymocytes
differentiate efficiently to DCs in response to multiple cytokines.
The relative expression of CD34 on thymocyte subsets is useful as a
marker for defining different stages of human thymic
development.23,24 The most primitive progenitors in the
human fetal thymus have been identified as
CD34brightCD1 thymocytes that express
low surface density CD38, but lack the T-lineage markers CD2 and CD5,
whereas the equivalent population in the postnatal thymus displays a
more mature CD2+CD5+CD38+
phenotype. In addition, fetal progenitors express low surface density
CD33 and intermediate levels of CD13 and HLA-DR, whereas cells with
this phenotype represent only 20% to 30% of
CD34+CD1 postnatal thymocytes (0.037% ± 0.011% of total thymocytes).14,20 Flow cytometry
analysis in Fig 1 shows that such cells
reside within the population of CD34+CD1
postnatal thymocytes that display the highest levels of CD34 and also
of CD44, suggesting that they represent the most immature progenitors
in the postnatal thymus. Conversely, thymocytes with a lower expression
of CD34 and CD44 (Fig 1) lack CD33 and display lower levels of CD13 and
HLA-DR. Despite these phenotypic differences, both cell subsets
(hereafter referred to as CD34+CD33lo and
CD34+CD33, respectively) were similar in
terms of CD2, CD5, and CD38 expression, although a slightly lower
expression of these markers was consistently found in the former. In
addition, CD34+CD33lo thymocytes displayed
higher levels of CD7 and CD45RA (Fig 1).
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| Fig 1.
Three-color flow cytometry analysis of postnatal
CD34+ CD1 thymocytes.
CD34+CD1 thymocytes isolated as described
in the Materials and Methods were analyzed by flow cytometry for the
correlated expression of CD44, CD34, and one of the indicated MoAbs.
Electronic gates were set as shown in the upper biparametric plot to
analyze the expression of CD2, CD5, CD38, CD7, CD33, CD13, HLA-DR, and
CD45RA antigens on CD44brightCD34bright
(unshaded areas) and CD44intCD34int (shaded
areas) thymocytes. Background values (dashed histograms) were
determined with isotype-matched irrelevant antibodies.
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To assess whether the CD34+CD33lo and the
CD34+CD33 thymocytes represent
sequential developmental stages within the postnatal CD34+CD1 thymic compartment, we sought
to determine their intrinsic differentiative capacity. In particular,
both isolated cell subsets were compared for their T/DC precursor
potential in culture, because unfractionated CD34+CD1 thymocytes cultured with IL-7
have been shown to produce simultaneously T-lineage cells (80% to
90%) and DCs (10% to 20%).14 However, in our initial
studies (Table 1), we observed that cell proliferation was greatly
improved and DC production was highly increased, mostly at the expense
of T-lineage cells (not shown), when IL-7 was used together with
various cytokines, namely IL-1, IL-6, SCF, and GM-CSF, used to
generate DCs in culture by others.5,25,26 Thus,
CD34+CD33lo and
CD34+CD33 thymocytes sorted as shown in
Fig 2A were cultured with the optimized mixture of cytokines, and their respective progenies were analyzed by
flow cytometry. As previously shown with unfractionated
CD34+CD1 thymocytes,14 cells
expressing upregulated levels of CD44 arised within 2 to 3 days from
both the CD34+CD33lo and
CD34+CD33 cultures, although in
different proportions. These were large cells (mean forward scatter,
554 ± 30) that had gradually lost surface CD5 to become
CD44brightCD5lo/. Interestingly, cells
with these morphologic and phenotypic features represented the major
cell progeny (80% to 90% by day 5) in all cultures initiated with
CD34+CD33lo thymocytes. The remaining
population (10% to 20%) was composed of small-sized cells (mean
forward scatter, 285 ± 15) that, as expected from T-lineage
precursors,14 displayed downregulated levels of CD44 but
retained CD5 expression. The opposite situation was found in the
CD34+CD33 cultures, in which a minor
fraction of cells (<25%) displayed the
CD44brightCD5lo/ phenotype, whereas most
of them (>75%) were CD44loCD5+. As shown in
Fig 2C, three-color analysis showed that the CD44bright and
the CD44lo cell progenies were significantly different in
terms of CD33 expression. CD44bright cells expressed
upregulated CD33 levels, whereas CD44lo cells were
CD33. Both populations were shown to keep relatively
high CD34 levels during the initial 3 to 4 days of culture (Fig 2C),
but thereafter they downregulated gradually CD34 expression (see
below), suggesting that they represent progenitor cells downstream from
the earliest CD34+CD33lo thymocytes.
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| Fig 2.
CD34+CD33lo and
CD34+CD33 postnatal thymocytes cultured
with multiple cytokines give rise to phenotypically distinct cell
progenies. (A) Sorted CD34+CD33lo and
CD34+CD33 postnatal thymocytes were
reanalyzed for the expression of CD34 and CD33. (B) The correlated
expression of CD44 and CD5 was independently analyzed on sorted
CD34+CD33lo (left panels) and
CD34+CD33 (right panels) cells either
before (day 0) or after 3 and 5 days of culture with a mixture of IL-7,
IL-1, IL-6, SCF, and GM-CSF. (C) The correlated expression of CD34
and CD33 was analyzed on electronically gated CD44bright
(left panel) and CD44lo (right panel) cell progenies
recovered at day 3 from the multicytokine-supported cultures of
CD34+CD33lo thymocytes.
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Kinetic studies showed that the small-sized
CD34+CD44loCD5+CD33
progeny differentiated within 6 to 8 days into
CD34CD1+CD4+CD8+
double-positive immature thymocytes, indicating that they represent T-lineage precursors (data not shown). These cells died in culture by
day 10. In contrast, essentially all
CD34+CD44brightCD5lo/CD33+
cells had differentiated by day 10 into large non-T-lineage cells lacking CD34 (data not shown) and expressing high levels of CD33, CD4,
CD40, and HLA-DR (Fig 3). These cells
showed a variable expression of CD1, although about 60% of them were
CD1bright. In all experiments, cells with this phenotype
had downregulated the CD7 molecule, but had acquired very high levels
of CD13, thus becoming homogeneously
CD13brightCD7lo/. In addition, a
variable proportion coexpressed CD11c, CD80, CD86, and HLA-DR, -DP, and
-DQ (Fig 3). Although no markers of mature T (CD3), B (CD19, CD22), and
NK (CD56) cells were found on these cells, the myeloid-related markers
CD14 and CD11b were detected at low and high expression levels,
respectively, on a variable proportion of them (data not shown). Cells
with this antigenic profile were irregularly shaped, displayed long
membrane processes, and behaved as professional antigen presenting
cells in a mixed lymphocyte reaction (Márquez et al14
and data not shown). Interestingly, these cells represented the only
cell progeny recovered from the CD34+CD33lo
cultures by day 10 (Fig 3). Therefore, the optimized mixture of
cytokines used in this study induces CD34+
CD33lo intrathymic precursors to generate CD34+
CD44bright CD5 lo/- CD33+
intermediate progenitors that differentiate exclusively into cells with
the phenotypic, morphological, and functional features associated with
DCs.27-29 Together, our data suggest that the
CD34+ CD33lo and CD34+
CD33 thymocyte precursors differ markedly in their
ability to generate DCs. In terms of absolute cell numbers,
CD34+ CD33lo thymocytes generate up to 10 times
more DCs than CD34+ CD33 thymocytes.
Therefore, CD34+ CD33lo postnatal thymocytes
are capable of serving as efficient DC precursors, while
CD34+ CD33 thymocytes seem to display a
reduced DC precursor potential but an increased fitness to generate
T-lineage cells.
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| Fig 3.
CD34+CD33lo thymocytes develop
efficiently into DCs in multicytokine-supported cultures.
CD34+CD33lo thymocytes were cultured for 10 days with the cytokine mixture described in Fig 2. Biparametric
histograms on the left show the correlated expression of CD44 and CD33,
CD44 and CD4, CD44 and CD1, CD40 and CD33, and CD7 and CD13 on the
cultured cells. Background fluorescence values were set by use of FITC-
and PE-conjugated isotype-matched irrelevant antibodies. Monoparametric
histograms on the right show the expression of HLA-DR, -DP, and -DQ,
CD11c, CD80, and CD86 (shaded histograms) on the same cultured cells. Background fluorescence (unshaded histograms) was determined by staining with isotype-matched irrelevant MoAbs plus PE-conjugated goat
antimouse Igs.
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CD34+CD33lo postnatal thymocytes display
NK cell precursor potential.
We next investigated whether CD34+CD33lo
thymocytes represent oligopotent progenitors able to generate not only
DCs, but other non-T-lymphoid cells such as NK cells.
Differentiation of CD34+CD33lo thymocytes into
cells with the characteristic
CD34CD56+CD3
phenotype of mature NK cells was observed in all experiments in which
IL-2 was added to the optimized cytokine mixture used for the
generation of DCs. As shown in Table 1, such culture conditions also
supported the production of DCs, so that both NK cells and DCs were
produced at equivalent numbers. Indeed, cells recovered by day 8 comprised a mixed population with two reciprocal phenotypic patterns
(Fig 4B). About 50% of these cells displayed the CD13brightCD7lo/ phenotype
associated with DCs, whereas the other 50% showed high CD7 expression
but low to undectetable levels of CD13. Electronic gates set
independently on each cell subset showed that, as expected for DCs,
CD13brightCD7lo/ cells expressed both
CD1 and CD4 and lacked CD56. In contrast, CD13lo/CD7+ cells were homogeneously
positive for the NK cell marker CD56, but lacked CD1 and CD4 (Fig 4B).
Thus, CD34+CD33lo thymocytes cultured with the
cytokine mixture plus IL-2 are able to generate simultaneously both DCs
and NK cells. Because no NK cells were formed in the absence of
IL-2, these data suggest a strong dependence on IL-2 by these
precursors for the development of NK cells. However, IL-2 by itself was
unable to induce the generation of NK cells, a common feature of all
cytokines included in the mixture when used singly (Table 1 and data
not shown). It did not induce the proliferation or support
the viability of CD34+CD33lo cells (Table 1),
indicating that NK cell development requires a broad spectrum of
cytokines in addition to IL-2. A striking finding was that
the majority (~90%) of cells recovered by day 4 from these cultures
were large-sized cells that displayed the CD34+CD44brightCD5lo/CD33+
non-T-lineage phenotype of large-sized DC precursors identified in
cultures lacking IL-2 (Fig 4A).
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| Fig 4.
CD34+CD33lo thymocytes develop
simultaneously into NK Cells and DCs in multicytokine-supported
cultures containing IL-2. CD34+ CD33lo
thymocytes were cultured with the mixture of IL-7, IL-1, IL-6, SCF,
and GM-CSF described in Fig 2, plus IL-2. (A) Depicts the correlated
expression of CD44 versus CD5, CD44 versus CD34, and CD34 versus CD33
at day 4. CD34+CD44bright
CD5lo/ cells represent 90% of total cells. High surface
CD33 expression was observed on 75% of total cells. (B) Shows the
phenotype of the cellular progeny at day 8. Cells were analyzed for the
correlated expression of CD7, CD13, and either CD1, CD4, or CD56.
Monoparametric histograms show the expression of CD1, CD4, and CD56
(shaded areas) on the CD7+CD13lo/ and
CD7lo/CD13+ cell subsets electronically
gated as shown in the biparametric plot. Background fluorescence
(unshaded histograms) was determined by staining with isotype-matched
irrelevant MoAbs plus PE-Cy5-conjugated goat antimouse Igs.
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Differentiation kinetics of thymic NK cells and DCs derived from
CD34+CD33lo intrathymic precursors.
Identical proliferation kinetics were observed in
multicytokine-supported cultures of CD34+CD33lo
thymocytes either containing or lacking IL-2. As shown in
Fig 5A, CD34+CD33lo
precursors grew exponentially, with a 30-hour doubling time, during the
initial (5 to 6 days) period of culture, resulting in a 15-fold
increase of cell numbers by day 6. Thereafter, cell expansion
decreased, but cell viability was maintained (>99%), and cell
numbers remained essentially unchanged from day 6 to day 10 through 15. Cellular recovery declined steadily afterwards, although viable cells
could be observed for up to 2 to 3 weeks. Thus, IL-2 had no effect on
the proliferation induced by the combination of IL-7, IL-1, IL-6,
SCF, and GM-CSF on CD34+CD33lo precursors (Fig
5A).
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| Fig 5.
Growth and differentiation kinetics of NK cells and DCs
derived from CD34+CD33lo thymocytes. (A)
CD34+CD33lo thymocytes (105/well)
were cultured during 9 days in 0.2 mL of medium supplemented with the
mixture of IL-7, IL-1, IL-6, SCF, and GM-CSF either without IL-2
( ) or with IL-2 ( ). An additional culture was set up without
IL-2, and IL-2 was added at day 4 ( ). The number of total viable
cells recovered at the indicated days was determined by trypan blue dye
exclusion. (B) CD34+CD33lo thymocyte cultures
set up as described in (A) were analyzed by flow cytometry for the
correlated expression of either CD7, CD13, and CD56, or CD7, CD13, and
CD1, at the indicated days. The data show the absolute numbers of
CD7+CD13lo/CD56+ NK cells
(open symbols) and
CD7lo/CD13+CD56 DCs (solid
symbols) recovered from cultures lacking IL-2 (,  ) or
supplemented with IL-2 either from day 0 (,  ) or from day 4 (,
 ).
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Microscopic inspection of cultures showed that, as reported for
precursors cultured only with IL-7,14
CD34+CD33lo cells growing in
multicytokine-supported cultures either with or without IL-2 became
aggregated after 24 hours to form large colonies containing greater
than 50 cells/colony. Such a colony formation was highly dependent on
cell density. Cells arising under both culture conditions were
morphologically indistinguishable during the initial 4 to 5 days. From
day 6 on, cells displaying a typical DC morphology were evident in both
cultures. However, these were the only cells arising in cultures
lacking IL-2, whereas DCs together with regularly shaped blastic cells
represented the end-product cells in cultures supplemented with IL-2.
No specific NK cell or DC maturation patterns were evident during the
initial (4 to 5 days) period of cellular expansion that was
characterized by the gradual loss of surface CD34. As shown in Fig 5B,
cells with the CD13brightCD7lo/
phenotype associated with DCs were first detectable in both culture conditions by day 5, whereas CD56+ NK cells arose 2 days
later, but exclusively in IL-2-supplemented cultures. Thereafter, the
absolute numbers of these two cell types increased gradually in their
respective cultures and peaked at day 9. Because total cell numbers
remained essentially unchanged from day 5 through 9, final
differentiation into cells with the immunophenotypic features of mature
DCs and NK cells may occur independently of cell proliferation.
Thymic DCs and NK cells develop simultaneously through a common
CD34+
CD44brightCD5lo/CD33+
intermediate stage.
Our kinetic studies provided evidence that the generation of
CD56+ NK cells in IL-2-supported cultures was accompanied
by a proportional decrease in the absolute numbers of DCs as compared
with numbers obtained from cultures lacking IL-2 (Fig 5B). This
indicates that NK cell production occurred at the expense of DCs.
Further support for this notion comes from additional studies in which
generation of CD56+ NK cells was also observed when
addition of IL-2 to the cytokine-supported cultures was delayed up to
day 4. In this last condition, NK cells appeared with delayed kinetics,
and the net NK cell production was reduced, whereas the absolute
numbers of DCs increased proportionally (Fig 5B). Thus, our data
indicate that IL-2 is dispensable for the initial generation of NK cell
precursors. Moreover, they suggest that the cytokine mixture without
IL-2 supports the generation of intermediate progenitors able to
produce both DCs and NK cells. Based on our previous data, we reasoned
that such progenitors might reside within the population of
CD34+CD44bright
CD5lo/CD33+ large cells representing the
immediate progeny arising from CD34+CD33lo
thymocytes cultured both with and without IL-2 (Figs 2 and 4). To
address this point, cells recovered at early time points (day 2) from
cultures lacking IL-2 were analyzed for their developmental potential
upon reculture for 5 additional days with the cytokine mixture, either
containing or lacking IL-2. In particular, we followed the kinetics of
CD7 and CD13 expression on ellectronically gated CD44bright
large cells arising under both culture conditions, because those markers were shown to define reciprocal phenotypic patterns on mature
NK cells and DCs (Fig 4B). At day 2, the CD44bright
large-sized progeny, gated as shown in Fig
6A, displayed a homogeneous CD7+CD13+ phenotype
that was maintained during the initial 4 days of culture, regardless of
the presence of IL-2. Thus, as shown in Fig 6B, the
CD44bright large cells recovered from both cultures at day
3 displayed an overlapping CD7+CD13+ phenotype.
Afterwards, CD44bright cells with upregulated CD13 and
downregulated CD7 surface levels increased gradually in cultures
lacking IL-2, giving rise to a homogeneous
CD13brightCD7lo/ DC progeny by day 7. In
contrast, a mixed population of CD44bright cells showing
opposite CD7 and CD13 expression patterns was evident by day 5 in
cultures containing IL-2. These cells segregated into two different
cell populations: one showing the characteristic CD13brightCD7lo/ DC phenotype and the
other expressing upregulated levels of CD7 but low to undetectable
surface CD13 by day 7. As expected from our previous data, day 7 CD13lo/CD7+ cells had acquired the CD56
NK cell marker (Fig 7). Taken together, our
data provide evidence that the
CD34+CD44brightCD5lo/CD33+
cells represent intermediate progenitors common to NK cells and DCs.
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| Fig 6.
NK cells and DCs develop simultaneously through a common
CD44bright intermediate stage. Cells derived from
CD34+CD33lo thymoctes cultured with IL-7,
IL-1, IL-6, SCF, and GM-CSF were recovered at day 2 and cultured for
5 additional days with the same cytokine mixture either with or without
IL-2. (A) Shows CD44 expression versus cell size (FS, forward scatter;
left plot) and the correlated expression of CD7 and CD13 on
electronically gated CD44bright large (forward scatter
>350) cells (right plot) at day 2. (B) Shows the kinetics of CD7 and
CD13 expression on electronically gated CD44bright large
cells recovered at the time points indicated after reculture with the
cytokine mixture either with or without IL-2.
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| Fig 7.
Induction of NKR-P1A expression on cultured
CD34+ CD33lo thymocytes. Cultures of
CD34+ CD33lo thymocyes were set up as
described in Fig 6. The correlated expression of NKR-P1A and CD13 was
analyzed by flow cytometry on electronically gated
CD44bright large cells at the indicated time points.
Percentages of NKR-P1A+ CD13+ cells
recovered at days 3, 4, 5, and 7 were 27%, 22%, 7%, and 5%,
respectively, in cultures lacking IL-2; and 29%, 40%, 30%, and 40%,
respectively, in cultures containing IL-2. The correlated expression of
NKR-P1A and CD56 was analyzed on electronically gated
CD44bright large cells recovered under both culture
conditions at day 7. Background fluorescence was determined by
sequential staining with isotype-matched (IgG1) irrelevant
MoAbs, FITC-conjugated goat antimouse IgG1, and
PE-conjugated isotype-matched irrelevant MoAbs.
|
|
NK cell progenitors derived from
CD34+CD33lo thymocytes can be identified
by the expression of the NKR-P1A receptor.
It has recently been shown that expression of the NKR-P1A surface
receptor in the absence of the CD56 NK cell marker defines an immature
pre-NK cell stage.21,30 Thus, kinetic studies on the
expression of the NKR-P1A molecule were then performed to assess
whether NKR-P1A+ intermediate NK cell precursors were
produced in our cultures. As shown in Fig 7, NKR-P1A was expressed on
25% to 30% of CD13+ cells recovered after 3 days from
cultures both with and without IL-2. These NKR-P1A+ cells
decreased gradually from day 4 in cultures lacking IL-2, so that by day
7 essentially all cells were NKR-P1A and displayed
the CD13bright (CD7lo/) DC phenotype.
However, in the presence of IL-2, increasing NKR-P1A expression levels
were detected throughout culture on a fraction of CD13+
cells that gradually downregulated surface CD13 to become
NKR-P1A+CD13lo/. All these cells
displayed a mature CD56+ phenotype by day 7 (Fig 7).
These data confirm that IL-2 is dispensable for the initial generation
of NKR-P1A+CD56 NK cell progenitors,
although it is required for their progression into
NKR-P1A+CD56+ mature NK cells. Related to this
point, it is worth noting that, regardless of the presence of IL-2, the
cytokine combination used in our studies induced the expression of CD25
(IL-2R) within the first 24 hours of culture. Such culture
conditions also supported the induction of CD122 (IL-2R  ), which
was first detectable 2 days later (day 3), coincident with the
appearance of NKR-P1A (Fig 8), and reached
a maximal expression by day 4. Thus, NK cell progenitors may become
sensitive to IL-2 only after they reach the NKR-P1A+ stage
of differentiation, at which they express both CD25 and CD122.
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| Fig 8.
Induction of CD25 (IL-2R) and CD122 (IL-2R)
expression on cultured CD34+CD33lo
thymocytes. CD34+CD33lo thymocytes cultured
for 1 or 3 days with IL-7, IL-1, IL-6, SCF, and GM-CSF were analyzed
for the correlated expression of CD34 and CD25 or CD34 and CD122.
Background fluorescence was determined by sequential staining with
isotype-matched irrelevant MoAbs, FITC-conjugated goat antimouse Igs,
and PE-Cy5-conjugated isotype-matched irrelevant MoAbs.
|
|
Frequency of DC- and NK cell-producing progenitors.
The overall differentiative capacity of CD34+
CD33lo thymocytes suggested that we had identified culture
conditions able to support the simultaneous generation of NK cells and
DCs with similar efficiencies. To directly analyze this possibility, we
sought to determine the frequency of DC and NK cell progenitors in
CD34+ CD33lo thymocytes by limiting dilution
assays. Thus, cells were plated at 300, 100, 33, 10, 3, and 1 cell per
well and cultured with the cytokine mix plus IL-2 for 16 days. At this
time, all wells were microscopically examined for the presence of live
cells expressing CD56 (NK cells) or lacking CD56 but showing a typical
dendritic morphology (DCs). The nature of such NK cells and DCs was
confirmed by carefully analyzing the composition of wells plated at
high cell concentrations (300 and 100 cells per well). As shown in Table 2 (experiment no. 1), the maximum
likelihood estimate of clonogenic precursors was 0.033 (1/30),
calculated with the single hit Poisson model.22 The
frequencies of DC and NK cell clonogenic precursors in this assay were
quite similar (0.031 [1/32] and 0.028 [1/35], respectively).
Interestingly, a substantial fraction of the positive wells seeded
under clonal conditions contained both type of cells, indicating that
they were seeded with bipotential DC/NK progenitors. However, the
individual DC and NK cell precursor frequencies obtained were lower
than expected considering the rapid increase in cell counts recorded in
high-density cultures. This suggested that proliferation and DC/NK cell
production would be favored by cell-cell contacts occurring in dense
cultures. As shown in Table 2, we found that the frequency of
clonogeneic precursors was about 20- to 30-fold higher when cells were
plated by limiting dilution after short-term (3 to 4 days) high-density cultures. In addition, these experiments allowed us to compare the DC
and NK cell precursor frequencies of the cell progeny generated in the
absence of IL-2 upon reculture with or without IL-2. Thus, cells
arising at day 3 (experiment no. 2) or day 4 (experiment no. 3) from
CD34+CD33lo multicytokine-supported cultures
lacking IL-2 (88% and 90% CD34+CD44bright,
respectively, <1% CD56+) were plated by limiting
dilution and cultured for 16 additional days with the cytokine mixture
either with or without IL-2. As shown in Table 2, the maximum
likelihood estimate of clonogenic precursors in the absence of IL-2 was
0.909 (1/1.10) and 0.952 (1/1.05) in experiments no. 2 and 3, respectively. All positive wells in these assays contained exclusively
DCs, indicating that close to 100% of cells recovered from short-term
cytokine-supplemented cultures lacking IL-2 can develop into DCs.
Interestingly, when the same cell samples were supplemented with IL-2,
25% (1/4) and 14% (1/7) of cells (in experiments no. 2 and 3, respectively) developed into NK cells, indicating that a significant
proportion of them were bipotential DC/NK cell precursors. The
estimated frequencies of such bipotential clones in experiments 2 and 3 was 19% (1/5.3) and 14% (1/7), respectively. Analysis of wells seeded
under clonal conditions in experiment no. 2 showed that, from a total
of 12 wells containing NK cells (of 60 plated), 10 wells contained also
DCs.
Identification of
CD34+CD44brightCD5lo/CD33+
intermediate thymocytes in vivo.
Flow cytometric studies were finally performed on ex vivo-isolated
thymocytes to investigate whether
CD34+CD44brightCD5lo/CD33+
progenitors, equivalent to the NK/DC intermediate precursors generated
in vitro under our culture conditions, could be identified in the human
postnatal thymus. To this end, thymocyte preparations depleted of
mature T, B, NK, and myeloid cells (lin) were
analyzed by flow cytometry after removal of early
CD34int-bright thymic progenitors, as described in the
Materials and Methods. As shown in Fig 9,
most thymocytes (~75%) within the remaining population were
CD44lo/ cells, whereas only up to 25% of them
expressed high levels of CD44. Both the CD44lo/ and
the CD44bright cell subsets were mostly composed of
CD34 cells, but also included a variable proportion
of cells expressing low but readily detectable surface CD34 (30% and
40% of CD44lo/ and CD44bright
thymocytes, respectively, in this experiment). Electronic gates set
independently on the CD34+ CD44lo/ and
the CD34+CD44bright populations showed that
thymocytes within the former subset displayed a
CD5+CD33CD13
T-lineage phenotype. In contrast,
CD34+CD44bright cells were homogeneously
CD33+CD13+, and most of them (>70%)
displayed low to negative CD5 surface expression. No expression of the
NKR-P1A receptor could be detected on cells within any cell subset
(data not shown). According to our results in vitro,
CD34+CD44brightCD5lo/CD33+CD13+
thymocytes identified in vivo were large-sized cells (mean forward scatter, 656), whereas
CD34+CD44lo/CD5+CD33CD13
thymocytes were small-sized cells (mean forward scatter, 319; Fig 9).
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| Fig 9.
Identification of
CD34+CD44brightCD5lo/CD33
intermediate thymocytes in vivo. Lin
thymocytes depleted of the most immature
CD34int-bright precursors, as described in the
Materials and Methods, were analyzed by flow cytometry for the
correlated expression of CD44, CD34, and one of the indicated MoAbs.
Electronic gates were set as shown in the upper biparametric plot to
analyze either the cell size (mean forward scatter, FSC) or the
expression of CD5, CD13, and CD33 antigens (shaded histograms) on
CD34+CD44bright (gate I) and
CD34+CD44lo/ (gate II) thymocytes.
Background staining values (unshaded histograms) were determined with
isotype-matched irrelevant antibodies.
|
|
| |
DISCUSSION |
We have shown previously that CD34+CD1
human postnatal thymocytes cultured with IL-7 differentiate efficiently
to T cells and also produce DC, albeit with a fivefold to 10-fold lower
efficiency. Both developmental pathways progress through separate
intermediate progenitors of different cell size that lose surface CD34
and simultaneously acquire opposite CD44 expression
patterns.14 Human pro-T cells, like their mouse
counterparts, downregulate CD44 to give rise to small-sized
CD44lo pre-T cells,31 whereas upregulation of
CD44 identifies large-sized thymic progenitors developing into DCs.
These observations suggested that attainment of the
CD44bright stage may be intimately linked to commitment of
intrathymic precursors into non-T cells. A relevant question is whether
these CD44bright cells represent intermediate precursors
already committed to the DC lineage or, alternatively, they include
cells able to form other thymic non-T cells such as NK cells. This has
important implications regarding the currently accepted premise that
the branching of human NK cells and T cells occurs downstream of the divergence of DCs.20 We have addressed this point in the
present study by analyzing the developmental relationship between DCs and NK cells derived in culture from a minor subset of
CD34+CD1 thymocytes that display the
highest levels of CD34 and may thus represent the most primitive
progenitors so far defined and isolated from the human postnatal
thymus. Such CD34bright postnatal thymocytes, like their
fetal counterparts,15,16 display a
CD33loCD13intHLA-DRint phenotype
equivalent to that of bone marrow CD34bright cells recently
identified as oligopotent progenitors common to all (T, B, NK, and
dendritic) lymphoid cell lineages.5 Therefore, it is likely
that the postnatal precursors analyzed in this study (referred to as
CD34+CD33lo) correspond to the initial bone
marrow-derived lymphoid progenitors that seed the postnatal thymus and
give rise to more differentiated CD34+CD33 thymocytes. This possibility
is supported by the fact that isolated CD34+CD33lo precursors differentiate
preferentially to T-lineage cells in IL-7-supported cultures (Table 1
and data not shown), but can also derive DCs and NK cells when more
complex cytokine combinations are used, whereas CD34+
CD33 thymocytes have a diminished DC (present study)
and NK cell (not shown) precursor potential but display more immediate
T-cell precursor activity. Although the B-cell precursor potential of
the CD34+CD33lo precursors has not been
analyzed in this study, it is likely that these early progenitors, like
their mouse counterparts,19 are also able to form B cells.
An important aspect of our study was the development of a
multicytokine-supported cell culture system (namely IL-7, IL-1, IL-6, SCF, and GM-CSF) that induced CD34+CD33lo
precursors to differentiate efficiently to DCs, but not to T-lineage cells. Similar cytokine combinations without GM-CSF have recently been
reported by Saunders et al26 to induce CD4lo
mouse intrathymic progenitors to develop almost exclusively to DCs,
although the T/B/NK precursor potential of such CD4lo
intrathymic precursors has been extensively reported.9-13
Strikingly, addition of IL-2 to our cultures resulted in the
simultaneous production of NK cells and DCs in similar proportions, but
had no effect on the cellular yield, indicating that NK cell generation occurs at the expense of DCs. A more relevant finding was that greater
than 90% of cells recovered at day 3 to 4 under both culture conditions were large-sized cells that displayed the non-T
CD44bright phenotype. Therefore, upregulation of CD44 may
be a necessary step common to the development of both DCs and NK cells.
These CD44bright cells still expressed the CD34 molecule,
albeit at reduced levels, indicating that they were relatively
primitive progenitors, but they had acquired high CD33 and CD13 surface
density. In contrast, the low proportion (<10%) of small-sized
CD44lo T-lineage progenitors arising at early time points
in our cultures displayed a
CD34+CD33CD13lo/
phenotype. It is worth noting that both the CD44bright and
the CD44lo cell subsets were identified in vivo,
thus providing additional support for their physiological
relevance as intermediate progenitors in the human postnatal thymus.
Although we cannot at present determine whether the
CD44bright cells still retain the capacity to form T cells,
this possibility seems very unlikely considering that progression to
the CD44bright stage results in the downregulation of the
T-cell-associated antigen CD5, whereas T-cell progenitors go through a
reciprocal CD44loCD5+ stage. Such a
differential expression of CD5 has proved particularly useful to trace
T-cell and NK cell lineage development from a subset of
CD34+CD5+CD33+CD13+
fetal thymocytes shown to display bipotential T/NK precursor activity.15 These data had been taken as evidence that,
once CD5 is lost from the cell surface, intrathymic precursors are no
longer able to form T cells, but rather they display NK cell precursor
potential. However, it remains to be determined whether the
CD5 NK cell progenitors previously
described15 are able to produce DCs as well.
We provide evidence that the CD34+CD44bright
progeny arising by day 3 to 4 under the cytokine-supported culture
conditions used in this study is a homogeneous DC progenitor population
(close to 100% clonogenic), able to form both DCs and NK cells upon
addition of IL-2, and propose that such CD44bright cells
include bipotential DC/NK cell progenitors. Interestingly, we found
that progression of such progenitors to the NK cell pathway could be
followed by the expression of the NKR-P1A receptor before acquisition
of the CD56+ mature NK cell phenotype, indicating that
these cells represent the thymic counterparts of a pre-NK cell
developmental stage recently identified in vivo in human
thymus21 as well as in adult and neonatal circulating
lymphocytes.30 Similarly, pre-NK cell precursors have
recently been identified in the mouse fetal thymus by the expression of
a different member of the NKR-P1 gene family, NKR-P1C.32
Although the minimal cytokine requirements for the generation of NK
cells have not been addressed in this study, our results support the
notion that IL-2 is dispensable for the production of
CD44brightNKR-P1A+CD56
intermediates with NK cell precursor potential. However, it is required
for the progression of NKR-P1A+CD56
precursors to NKR-P1A+CD56+ mature NK cells.
Thus, the effects of IL-2 in our NK cell differentiation system appear
to be stage-specific. This supports the current view that NK cell
progenitors at distinct developmental stages display differential
cytokine requirements: the most mature NK precursors respond readily to
IL-2, whereas more immature progenitors need complex mixtures of
cytokines and/or stromal cells.33-36 It is worth
noting that IL-15, a cytokine known to play a role in NK cell
differentiation, has recently been reported to display similar
stage-specific effects,36-38 this probably reflecting the fact that both IL-2 and IL-15 use the and chains of the IL-2R. This concurs with our observation that, as shown for bone marrow CD34+ NK cell progenitors,34 the thymus-derived
NK cell progenitors identified in this study become sensitive to IL-2
only after they reach the stage of differentiation at which they
express the IL-2R (CD122) complex. Given the coincident
expression kinetics of IL-2R and NKR-P1A, it can be speculated
that signaling through the IL-2R complex is the key molecular
event that determines the final developmental fate of
NKR-P1A+ precursors. In addition, the finding that
expression of the IL-2R (CD25) precedes that of the IL-2R,
together with the fact that low doses of IL-2 promote efficient NK cell
production, may suggest that IL-2 responsiveness in our cultures
involves the high-affinity IL-2R. However, it is likely that,
during the process of intrathymic NK cell development in vivo, IL-15
that is efficiently produced by the thymic epithelium,37
rather than IL-2, is the physiological ligand that binds the
IL-2R complex and promotes final differentiation of pre-NK cells
to mature NK cells.
Taken together, our results provide evidence that the intrathymic DC
and NK cell developmental pathways share a common transitional stage
that is morphologically and phenotypically distinct from the most
immature T-lineage precursors. However, recent results in mice by Wu et
al19 indicate that there is a coincidence between the
thymic DC precursors and the earliest T-cell precursors. Because it was
shown in a previous study13 that the cellular precursors analyzed by Wu et al19 can also give rise to NK cells, such cells may thus represent tripotential T/DC/NK precursors. Although the
T-cell precursor potential of the human DC/NK precursor subset described in this study remains to be examined, we favor the concept that, once the B-cell potential is lost, intrathymic NK cells and DCs
branch off the T lineage through a common intermediate progenitor. It
has to be stressed that, in support of this view, it has recently been
shown that postnatal T-cell precursors, but neither NK cells nor DCs,
are produced in mice mutant for the transcription factor Ikaros,
indicating that Ikaros is essential for establishing early branch
points into these two pathways from the main T-cell pathway in the
postnatal thymus.39 The simultaneous development of NK
cells and DCs may provide new insights into the physiological
mechanisms that control the MHC-dependent NK cell education process in
the thymus.40
| |
FOOTNOTES |
Submitted September 8, 1997;
accepted November 25, 1997.
C.M. and C.T. are joint first authors.
Supported in part by grants from Glaxo Wellcome S.A., Grants No.
SAF95-0006 and SAF97-0161 from Comisión Interministerial de
Ciencia y Tecnología (CICYT), and Grant No. FIS-94/0266 from the Fondo de Investigaciones Sanitarias. The Centro de Biología Molecular "Severo Ochoa" is partially supported by the
Fundación Ramón Areces. C.M. has a postdoctoral contract
from the Consejo Superior de Investigaciones Científicas
(CSIC)-Fundación Ramón Areces, and C.T., J.M.F., and A.R.R.
are fellows from FIS, Glaxo Wellcome S.A., and Ministerio de
Educación y Ciencia, respectively.
Address reprint requests to María L. Toribio, PhD, Centro de
Biología Molecular "Severo Ochoa," Universidad
Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Drs J. Banchereau, J.C. Gutiérrez-Ramos, I. Mérida, J. Bodmer, and E. Leonardo for the generous gift of antibodies; Dr A. Alvarez for assistance with cell sorting; and the
Pediatric Cardiosurgery Units from the Centro Especial Ramón y
Cajal and Ciudad Sanitaria La Paz (Madrid) for the thymus samples.
| |
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Abstract
Introduction
Abstract